This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gramnegative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/ alkali/heat lysis. The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert

Edible seaweed (Palmaria palmata) has traditionally been consumed rawafter harvesting from coastal shorelines around Northern Ireland.To date, there have been no reports examining the microbiology of this material, hence itwas the aimof this study to detect the diversity of themicro£ora found on readytoeat produce. Conventionalmicrobiological analyses of the product failed to detect any gastrointestinal pathogens and gave a mean total count of 1•3×105 cfug-1. 16S rRNA sequencing of culturable bacteria identified the Sanguibacter/Oerskovia/Cellulomonas complex, the Clavibacter/Frigoribacterium/urtobacterium complex, Enterobacter agglomerans, Erwinia herbicola, Flavobacterium spp., Micrococcus lylae, Microbacterium spp., Corynebacterium spp., and Dietza maris. A comparison of growth of isolated environmental organisms was performed to ascertain the most appropriate artificial culture media to employ for their culture in vitro. Tryptone soya broth with yeast extract (TSBYE) and brainfiheart infusion brothwith yeast extract (BHIYE) may be employed as suitable basal broth media for the laboratory culture of these organisms.This is the ¢rst preliminary report on the microbial diversity of edible seaweed and demonstrated the presence of several halophilic genera and species in fresh ready-to-eat edible seaweed from Northern Ireland. Although no gastrointestinal pathogens were cultured from this material, a larger study requiring examination of seasonal e¡ects, quality of marine water and e¡ect of drying on faecal pathogens, is required to support a functional HACCP-based approach to ensuring safety of this product.

Optimum detection of the Burkholderia cepacia complex (BCC) from sputum of patients with cystic fibrosis (CF) is essential in preventing patient-to-patient transmission of this organism. The aim of this study was to develop an improved PCR assay with reference to sensitivity for the direct detection of BCC organisms from CF sputum employing the recA locus. The sensitivity results of three recA PCR assays were compared using various combinations of previously published primers. These included (i) a single-round approach using the primer set BCR1/BCR2, yielding a 1036-bp product, (ii) a single-round approach using the primer set BCR1/Mr, yielding a 465-bp product, and (iii) a semi-nested PCR (SN-PCR) approach using the primer set BCR1/BCR2 followed by BCR1/Mr. The sensitivity of these assays were determined by spiking B. cepacia-free sputum with known numbers of four strains of BCC, namely, genomovar II [B. multivorans] (C1576), genomovar IIIa (C5424, C6433) and genomovar IIIb (C1394). Following optimization, the chosen assay was performed on 14 patients. Employment of the singleround assay with BCR1/BCR2 was the least sensitive with a detection threshold of 107 cfu/g sputum for GIIIa and GIIIb, and 108 cfu/g sputum for GII. Sensitivity was improved by targeting the smaller amplification region of the recA locus (465 bp) employing the BCR1/Mr primer pair, in combination with a single-round approach, whereby the detection threshold was improved by 1 log for each genomovar. Employment of the semi-nested assay demonstrated optimum sensitivity, whereby the detection threshold increased to 101 and 102 cfu/g sputum for genomovar IIIa/IIIb and genomovar II, respectively. Subsequent genomovar characterisation can be performed by sequencing of the PCR amplicon without the need for culture which may be beneficial in patients in the initial stages of colonisation or who are transiently colonised and who may be culture-negative for BCC.

B.C. MILLAR, J. E. MOORE, J. XU, M. J. WALKER, S. HEDDERWICK AND R. MCMULLAN. 2002. Aims: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates. Methods: Twenty-one invasive and 18 noninvasive isolates were examined by PCR amplification of a transposable intron region in the 25S rRNA gene. Isolates were genotyped following analysis of the size of resulting DNA amplicons. The isolates could be subdivided into four genotypes (A-D). Results: There was no significant difference between the frequency and genotype distribution of the invasive and noninvasive Candida isolates. Impact of the Study: Therapeutic prophylaxis against candidal infections remains an area of controversy. Any diagnostic markers that reflect the potential of isolates to become invasive should be fully explored, so that more focused antifungal intervention should be targeted at these patients with these potential invasive markers. This study demonstrated that analysis of the transposable intron region in the 25S rRNA gene may be useful in helping to differentiate C. albicans from C. dubliniensis isolates, without the need for sequence analysis, which may not be readily available at primary diagnostic laboratories. However, employment of this genotypic assay is not a suitable locus to determine invasiveness and other more reliable markers of invasiveness should be sought.